利迪链霉菌初级代谢关键基因的克隆与功能分析
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摘要
利迪链菌素是利迪链霉菌产生的一种结构全新、抑菌效果明显的新型抗生素,具有很高的潜在应用价值。然而利迪链菌素的产量低是其应用的限制因素,加强初级代谢途径,提高其产量是一条可行的途径。本文克隆了利迪链霉菌初级代谢的几个关键基因,并进行了功能分析。
研究了利迪链霉菌对木糖的利用能力,结果表明利迪链霉菌能利用木糖作为唯一碳源,但生长比较缓慢。在低浓度葡萄糖培养基上,木糖对生长具有显著促进作用。从利迪链霉菌中克隆到了木糖代谢的关键基因木酮糖激酶编码基因xylB,长度为1446bp,编码481个氨基酸。这表明,虽然利迪链霉菌利用木糖效率低,但仍然具有木糖代谢的遗传基础。
采用同源克隆策略,克隆了利迪链霉菌葡萄糖代谢途径中的4个基因。葡萄糖-6-磷酸脱氢酶基因zwf2,长度1542bp,编码513个氨基酸,在242-252位具有AXXXGXGGXA (可能的NADP+结合位点)结构域,43位具有保守残基K(可能的葡萄糖-6-磷酸结合位点)。葡萄糖激酶编码基因glkA,长度954bp,编码317个氨基酸。磷酸果糖激酶基因pfkA1,长度1026bp,编码341个氨基酸。XylB,GlkA,PfkA1属于糖激酶,基于广义酸碱催化机理分析,催化活性区域可能分别为PYLDGERT (326-333位), CXCGXGCXEXY (168-180),DIXXTDXTXTFGFD (125-140位)。异柠檬酸脱氢酶基因idh长2220bp,编码739个氨基酸,Idh催化活性区域可能是HLKATMM(254-260位)。
构建了zwf2组成型强表达载体,通过接合转移,将其转化到利迪链霉菌中。经过筛选鉴定,得到了重组利迪链霉菌。
通过以上糖代谢关键基因克隆与功能分析研究,并构建了zwf2高表达菌株,为以后深入研究初级代谢对利迪链菌素生物合成的影响奠定了基础。
Streptolydigin is a novel antibiotic produced by Streptomyces lydicus. It has high antibacteral activity with potential commercial value. But the low productivity limits its application. Enhancing primary metabolism is a feasible approach to improve the antibiotic production. In this study we cloned several key genes of primary metabolism and analyzed their functions.
Xylose significantly promoted mycellium growth rate when the media contained low concentration of glucose.The cloned xylB of xylose metabolism gene cluster is 1446 bp in length and encodes 481 amino acids. The results showed S. lydicus can utilize xylose though the efficiency is low.
Using the degenerated primers based on the conserved domains, we cloned four key genes (zwf2, glkA, pfkA1, idh) of glucose metabolism from S. lydicus. zwf2 is 1542 bp long and encodes a putative enzyme of 513 amino acids. glkA is 954 bp in length and encode 317 amino acids. pfkA1 is 1026 bp in length and encode 341 amino acids. idh is 2220 bp long and encodes a putative enzyme of 739 amino acids. The active domains of Zwf2 may be AXXXGXGGXA (putative NADP+ binding site) and lysine43 (glucose-6-phosphate binding site). The active domain of three sugar kinases, XylB, GlkA and PfkA1 with the general acid and alkaline catalytic mechanism, may be PYLDGERT, CXCGXGCXEXY and DIXXTDXTXTFGFD while Idh’s active domain may be the highly conserved domain HLKATMM. We constructed a constitutive expression plasmid pIB139-zwf2, and then transformed to the S. lydicus.
The cloning and function analysis of these key genes of primary metabolism and construction of zwf2-high expression strain lay a foundation of further study on the relationship between primary metabolism and streptolydigin biosynthesis.
研究了利迪链霉菌对木糖的利用能力,结果表明利迪链霉菌能利用木糖作为唯一碳源,但生长比较缓慢。在低浓度葡萄糖培养基上,木糖对生长具有显著促进作用。从利迪链霉菌中克隆到了木糖代谢的关键基因木酮糖激酶编码基因xylB,长度为1446bp,编码481个氨基酸。这表明,虽然利迪链霉菌利用木糖效率低,但仍然具有木糖代谢的遗传基础。
采用同源克隆策略,克隆了利迪链霉菌葡萄糖代谢途径中的4个基因。葡萄糖-6-磷酸脱氢酶基因zwf2,长度1542bp,编码513个氨基酸,在242-252位具有AXXXGXGGXA (可能的NADP+结合位点)结构域,43位具有保守残基K(可能的葡萄糖-6-磷酸结合位点)。葡萄糖激酶编码基因glkA,长度954bp,编码317个氨基酸。磷酸果糖激酶基因pfkA1,长度1026bp,编码341个氨基酸。XylB,GlkA,PfkA1属于糖激酶,基于广义酸碱催化机理分析,催化活性区域可能分别为PYLDGERT (326-333位), CXCGXGCXEXY (168-180),DIXXTDXTXTFGFD (125-140位)。异柠檬酸脱氢酶基因idh长2220bp,编码739个氨基酸,Idh催化活性区域可能是HLKATMM(254-260位)。
构建了zwf2组成型强表达载体,通过接合转移,将其转化到利迪链霉菌中。经过筛选鉴定,得到了重组利迪链霉菌。
通过以上糖代谢关键基因克隆与功能分析研究,并构建了zwf2高表达菌株,为以后深入研究初级代谢对利迪链菌素生物合成的影响奠定了基础。
Streptolydigin is a novel antibiotic produced by Streptomyces lydicus. It has high antibacteral activity with potential commercial value. But the low productivity limits its application. Enhancing primary metabolism is a feasible approach to improve the antibiotic production. In this study we cloned several key genes of primary metabolism and analyzed their functions.
Xylose significantly promoted mycellium growth rate when the media contained low concentration of glucose.The cloned xylB of xylose metabolism gene cluster is 1446 bp in length and encodes 481 amino acids. The results showed S. lydicus can utilize xylose though the efficiency is low.
Using the degenerated primers based on the conserved domains, we cloned four key genes (zwf2, glkA, pfkA1, idh) of glucose metabolism from S. lydicus. zwf2 is 1542 bp long and encodes a putative enzyme of 513 amino acids. glkA is 954 bp in length and encode 317 amino acids. pfkA1 is 1026 bp in length and encode 341 amino acids. idh is 2220 bp long and encodes a putative enzyme of 739 amino acids. The active domains of Zwf2 may be AXXXGXGGXA (putative NADP+ binding site) and lysine43 (glucose-6-phosphate binding site). The active domain of three sugar kinases, XylB, GlkA and PfkA1 with the general acid and alkaline catalytic mechanism, may be PYLDGERT, CXCGXGCXEXY and DIXXTDXTXTFGFD while Idh’s active domain may be the highly conserved domain HLKATMM. We constructed a constitutive expression plasmid pIB139-zwf2, and then transformed to the S. lydicus.
The cloning and function analysis of these key genes of primary metabolism and construction of zwf2-high expression strain lay a foundation of further study on the relationship between primary metabolism and streptolydigin biosynthesis.
引文
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[3] Rosen T, Fernandes P B, Marovich M A et al. Aromatic dienoyl tetramic acids. Novel antibacterial agents with activity against anaerobes and staphylococci. J Med Chem. 1989, 32: 1062–1069.
[4] Kyzer S, Zhang J, Landick R. Inhibition of RNA polymerase by streptolydigin: no cycling allowed. Cell. 2005, 122:494–496.
[5] Temiakov D, Zenkin N, Vassylyeva M N et al. Structural basis of transcription inhibition by antibiotic streptolydigin. Mol Cell. 2005, 19:655–666.
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[9] Chen H, Harrison P H. Investigation of the origin of C2 units in biosynthesis of streptolydigin. Org Lett. 2004, 6:4033–4036.
[10] Chen H, Olesen S G, Harrison P H.Biosynthesis of Streptolydigin: Origin of the Oxygen Atoms. Org Lett. 2006, 8:5329–5332.
[11] Parekh S, Vinci V A, Strobel R J. Improvement of microbial strains and fermentation processes. Appl Microbiol Biotechnol, 2000, 54:287–301.
[12] Tan T W, Zhang M, Wang B W et al. Screening of high lipase producing Candida sp. and production of lipase by fermentation, Process Biochem. 2003, 39: 459–465.
[13] Bai D M, Zhao X M, Li X G et al. Strain improvement of Rhizopus oryzae for over-production of l(+)-lactic acid and metabolic flux analysis of mutants. Biochem Eng J. 2004, 18:41–48
[14] Kumar M S, Kumar P M, Sarnaik H M et al. A rapid technique for screening of lovastatin-producing strains of Aspergillus terreus by agar plug and Neurospora crassa bioassay. J Microbiol Methods. 2000, 40:99–104.
[15] Li Q S, Wang L J, Li Y R. Color development with rational screening method for improved l-glutamate oxidase-producing strains. Enzyme Microb Tech. 1996, 18: 7–9.
[16] Kumar D, Garg S, Bisaria V S et al. Production of methionine by a multi-analogue resistant mutant of Corynebacterium lilium. Process Biochem. 2003, 38: 1165–1171.
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[18] Hatvani L, Manczinger L, Kredics L et al. Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion. Antonie Van Leeuwenhoek. 2006, 89(3-4): 387-393.
[19] Khattab A A, Bazaraa W A. Screening, mutagenesis and protoplast fusion of Aspergillus niger for the enhancement of extracellular glucose oxidase production. J Ind Microbiol Biotechnol. 2005, 32(7):289–294.
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